A parasitic nematode induces dysbiosis in susceptible but not resistant gastropod hosts

Abstract Animals’ gut microbiomes affect a wide array of biological processes including immunity and protection from pathogens. However, how the microbiome changes due to infection by parasites is still largely unknown, as is how the microbiome changes in hosts that differ in their susceptibility to parasites. To investigate this, we exposed two slug species of differing susceptibility to the parasitic nematode Phasmarhabditis hermaphrodita (Deroceras reticulatum is highly susceptible and Ambigolimax valentianus resistant to the nematode) and profiled the gut microbiota after 7 and 14 days. Before infection, both slug species’ microbiota was dominated by similar bacterial genera: Pseudomonas (by far the most abundant), Sphingobacterium, Pedobacter, Chryseobacterium, and Flavobacterium. In the resistant host A. valentianus, there was no significant change in the bacterial genera after infection, but in D. reticulatum, the bacterial profile changed, with a decrease in the abundance of Pseudomonadaceae and an increase in the abundance of Flavobacteriaceae and Sphingobacteriaceae after 7 days postinfection. This suggests nematode infection causes dysbiosis in hosts that are susceptible to infection, but the microbiome of resistant species remains unaltered. In summary, the regulation of the immune system is tightly linked with host survival, and nematode infection can alter the microbiome structure.

A host and its microbiome have complex interactions at many levels often undergoing co-evolutionary pressures, as such it is not correct to truly consider them separate entities. Instead, it is preferable to consider the host and its microbial community as a holobiont recognizing its diversity and dynamic association (Theis et al., 2016). Brealey et al. (2022) suggested that parasites can also be considered holobionts, as they are residing within the microbial community of their holobiont host. Brealey et al. demonstrated that infection by the intestinal cestode Eubothrium spp. is associated with dysbiosis of the Atlantic salmon gut microbiome. The cestode Eubothrium spp. was shown to select for bacteria belonging to the family mycoplasmas when infecting Atlantic salmon, this highlights the importance of considering the parasite holobiont when studying parasitic infections (Brealey et al., 2022).
Currently, most research is focused on vertebrate holobiont systems, yet invertebrate systems have the potential to become tractable laboratory models for holobiont research due to their relatively simple gut communities and ease of laboratory culture (Newton et al., 2013). Cardoso et al. (2012), Landry et al. (2015), and Jackson et al. (2021) investigated the effects of environment and diet on invertebrate microbiomes showing how the microbiome can shift due to external factors. Cardoso et al. (2012) demonstrated that the microbiome of the land snail Achatina fulica can be altered by their diet, with a high-sugar diet leading to an increase in the abundance of phyla Bacteroidota and Bacillota. Landry et al. (2015) discovered that the majority of bacteria found in the spruce budworm (Chroistoneura fumiferana) microbiome belonged to the phylum Pseudomonadota, and their experiments showed species diversity was significantly affected by environment and diet. Jackson et al. (2021) identified and determined that the core microbiome of the slug Ambigolimax valentianus can be influenced by diet (sterile and non-sterile) and environment (garden or lab-reared). It has also been shown that parasitic infections can alter the balance of the host's microbiome, causing dysbiosis (Brealey et al., 2022) but more research is needed to fully understand this effect. Furthermore, while many studies have concentrated on freshwater parasites and hosts there is little information about the role the gut microbiome plays in host immunity in terrestrial environments.
One such host/parasite system that could provide insight is that of slugs and their nematode parasites. Several slug species are global pests of crops, responsible for millions of pounds of damage each year (Nicholls, 2014). Slugs are parasitized by flies, trematodes, viruses, and microsporidia, but the most prevalent group is the nematodes (Wilson et al., 2004), with 108 species using gastropods as intermediate, definitive, and paratenic hosts (P. S. Grewal, Grewal, Tan, et al., 2003). One species, Phasmarhabditis hermaphrodita, is a lethal parasite that has been developed as a biological control agent (Nemaslug ® ) to control pestiferous slugs on gardens and farms in the UK and northern Europe by BASF Agricultural Specialities (R. Rae et al., 2007). Infective stage nematodes (Figure 1a) are applied to soil and are attracted to chemical cues such as slug mucus and feces (R. G. Rae et al., 2009;Wilson et al., 2006), they then enter through the back of the mantle proliferate, and kill the slug in 4-21 days (Tan and Grewal, 2001b;Wilson et al., 1993). Self-fertilizing adult nematodes ( Figure 1b) reproduce on the cadaver of the slug, and once the food supply is depleted, they develop into new infective juveniles where they search for new slug hosts in the soil. P. hermaphrodita has successfully been used to control slugs in many crops including lettuce (Wilson et al., 1995) and in floriculture, for example, orchids (S. K. Grewal, Grewal, Hammond, et al., 2003). P. hermaphrodita is not the only nematode to kill slugs, there are several other species, for example, P. californica and P. neopapillosa that can kill susceptible slugs (Sheehy et al., 2022). There is natural variation in the pathogenic ability of P. hermaphrodita (Cutler and Rae, 2020) and crucially, P. hermaphrodita (and other Phasmarhabditis species) are facultative parasites that can be cultured under laboratory conditions allowing infection experiments to be carried out (see Cutler and Rae, 2020). P. hermaphrodita has been shown to infect and kill several species of pestiferous slug (Rae et al., 2009;Wilson et al., 1993) though the mechanism of pathogenesis is debated. It was thought the nematodes vectored the bacterium Moraxella osloensis into the hemocoel of the slug, and this was responsible for host death (Tan and Grewal 2001a, 2001b, 2002. However, recent research by Sheehy et al. (2022) failed to find this bacterium in the next generation of P. hermaphrodita (and two other Phasmarhabditis species, P. californica, and P. neopapillosa) after killing a slug using 16S rRNA metagenomic sequencing (Sheehy et al., 2022). Furthermore, these authors showed using 16S rRNA amplicon sequencing that M. osloensis is a Psychrobacter spp. Therefore, the role bacteria play in causing death to slugs is currently unknown. This warrants further research as these are the only genera of the nematode to evolve to kill slugs and snails out of the entire Nematoda phylum (consisting of an estimated one million species).
We aimed to discover whether P. hermaphrodita affects the gut microbiota of two gastropod host species of differing susceptibility to the nematode. The highly susceptible slug Deroceras reticulatum ( Figure 1c), a common pest species with worldwide importance (Hutchinson et al., 2014), is killed by P. hermaphrodita in 4-21 days (Rae et al., 2009;Wilson et al., 1993) while Ambigolimax valentianus ( Figure 1d) is resistant to being killed by P. hermaphrodita (Dankowska, 2006;Ester et al., 2003). How and why these slug species differ so dramatically in their susceptibility to P. hermaphrodita is unknown. To investigate the potential role of the gut microbiome we used 16S SSU ribosomal DNA metagenomic profiling to track changes in the gut microbiome of each species before and after infection with P. hermaphrodita.

| Infection of gastropod hosts with P. hermaphrodita
We used a standard bioassay to infect D. reticulatum and A. valentianus with P. hermaphrodita (see Cutler and Rae, 2020;Sheehy et al., 2022). Briefly, infective stage P. hermaphrodita DMG0001 was added in doses of either 500 or 1000 nematodes in 2 mL of water to cotton bungs at the bottom of separate 20 mL universal tubes. Two adult slugs (either D. reticulatum or A. valentianus) were added to each tube, a cotton wool plug was placed on top, and the lid was loosely closed. The slugs were exposed for 7 days at 10°C in the dark, after which feces were collected using a pipette tip to transfer to a 2 mL Eppendorf tube for DNA extraction.

| Profiling the gut microbiota from feces of D. reticulatum and A. valentianus
Individuals were grouped as follows. Group 1 (five D. reticulatum fed a lab diet for 7 days); Group 2 (five D. reticulatum fed a lab diet for 14 days); Group 3 (five D. reticulatum fed a lab diet and infected on Day 7 with P. hermaphrodita with feces collected 7 days postinfection-14 days in total); Group 4 (three A. valentianus fed a lab diet for 7 days); Group 5 (three A. valentianus fed a lab diet for 14 days); Group 6 (three A. valentianus infected with P. hermaphrodita with feces collected 7 days postinfection-14 days in total). Feces were collected from each slug for DNA extraction.
DNA was extracted from feces using DNeasy PowerSoil Pro Kit (Qiagen) following the manufacturer's instructions. The presence of bacterial DNA was checked after extractions using PCR amplification of the hypervariable regions of the 16S rRNA gene. This was carried out using the primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and F I G U R E 1 The infective juvenile stage of Phasmarhabditis hermaphrodita (a) develops into self-fertilizing adults (b) that reproduce on a slug cadaver. P. hermaphrodita can infect and kill the susceptible slug species Deroceras reticulatum (c) but not the resistant slug species Ambigolimax valentianus (d).
For each OTU, QIIME (Version 1.7.0) in the Mothur method was performed against the SSU rRNA database of SILVA Database for species annotation at each taxonomic rank (Threshold:0.8~1) (Quast et al., 2012). MUSCLE (Version 3.8.31) (Edgar, 2004) was used to obtain the phylogenetic relationship of all OTUs.
OTUs abundance information was normalized using a standard of sequence number corresponding to the sample with the least sequences. OTUs were analyzed for Alpha diversity (Wilcoxon test function) and Beta diversity (AMOVA-Analysis of Molecular Variance) to obtain richness and evenness information in samples.
AMOVA was also used to compare the taxonomic compositions of infected and noninfected slugs in weighted PCoA. Analysis of Alpha and Beta diversity were all performed on the normalized data and calculated with QIIME (Version 1.7.0). Significant intragroup variation is detected via MetaStats based on their abundance. Group 6, infected with P. hermaphrodita with feces collected 7 days after infection. A core microbiome of 384 OTUs with just eight phyla represented but in similarity to D. reticulatum the core microbiome is dominated by bacteria from the phylum Psuedomonadota.
The core microbiomes for D. reticulatum and A. valentianus indicate several shared bacterial associations at the phylum level.
D. reticulatum and A. valentianus are associated with a wide range of bacteria from several phyla, including Pseudomonadota and Bacteroidota (Figure 3a,b).

| No effect of sustained laboratory-based diet on the microbiome of D. reticulatum and A. valentianus
The sustained laboratory-based diet does not lead to a significant difference in the microbiome of D. reticulatum or A. valentianus.
Group 1 (D. reticulatum) and Group 4 (A. valentianus) were fed a diet of lettuce and carrot for 7 days before feces collection. While Group 2 (D. reticulatum) and Group 5 (A. valentianus) were fed a diet of lettuce and carrot for 14 days before feces collection. Neither richness (alpha diversity analysis with Wilcoxon test function p > 0.5) nor microbiome structure (beta diversity analysis by AMOVA p > 0.1) showed a significant change due to the sustained laboratory diet. As shown in Figure 3a,b the relative abundance at the phylum level is similar between Group 1 and Group 2 for D. reticulatum and between Group 4 and Group 5 for A. valentianus. Therefore, the laboratorybased diet does not affect microbiome diversity in either slug species. Bacteria with significant intra-group variation were detected via MetaStats based on their abundance, through this analysis, a significant decrease in the abundance of Pseudomonadota in D. reticulatum hosts was seen after infection with P. hermaphrodita between Group 1 and Group 3 (p < 0.0005) and Group 2 and Group 3 (p < 0.002) (Figure 6a). This decrease specifically affected bacteria from the class of Gammaproteobacteria. Additionally, there are significant decreases (p < 0.0005) in the abundance of bacteria in F I G U R E 3 (a) Deroceras reticulatum associates with a wide range of bacteria from several phyla Psuedomonadota, Bacillota, Actinomycetota, and Bacteroidota. While the laboratory diet (Group 2) has minimal effect on the relative abundance of these phyla, infection with the malacopathogenic nematode Phasmarhabditis hermaphrodita affects the relative abundance of Pseudomonadota and Bacillota. (b) Ambigolimax valentianus associates with a wide range of bacteria from several phyla Psuedomonadota, Bacillota, and Actinomycetota. Neither the sustained laboratory diet nor the infection with P. hermaphrodita significantly affects the relative abundance of the bacteria associated with A. valentianus.

| DISCUSSION
In this study, we have determined that bacteria from the phyla Pseudomonadota, Bacteroidota, and Actinomycetota were abundantly present in the core microbiomes of D. reticulatum and A. Both species showed a large abundance of Pseudomonadota before infection, this abundance of Pseudomonadota was also seen in the work from Cardoso et al. (2012) on the microbiome of the giant land snail Lissachatina fulica. Though Cardoso also showed that when the land snail was fed a diet rich in sugarcane, there was a greater abundance of Bacteroidota and Bacillota (Cardoso et al., 2012), demonstrating the malleability of the microbiome in response to external forces, in this instance diet.
We also investigated whether and how infection with P.
hermaphrodita affected the gut microbiota of two host gastropod species. Whilst susceptible host species D. reticulatum showed a significant shift in the microbiome, in particular, the abundance of Pseudomonadota and Bacteroidota, the resistant species A. valentianus showed no such effect.
Further investigation is still required to understand whether the parasitic nematode directly alters the microbiome of the slug or if the microbiome is shifting as a secondary effect of the ill health of the slug host during a nematode infection. There is evidence in other host/parasite systems to show parasites can directly affect their host's microbiome (Gaulke et al., 2019;Reynolds et al., 2015;Walk et al., 2010). Walk et al. (2010)  Furthermore, infection with whipworm (Trichuris muris) reduces microbiome alpha diversity in mice (Houlden et al., 2015) but can promote the growth of Lactobacillus (Holm et al., 2015). Houlden et al. (2015) also showed that the presence of the parasite is required to maintain the shift in the microbiota as once the parasite is removed the microbiota transitions back to that of an uninfected animal, suggesting that the parasite is directly changing the microbiome of the host rather than a microbiome shift being an indirect response. In addition to the parasites changing the microbiota of the host, White et al. (2018) found the parasitic nematode T. muris acquired microbiota from its mouse host, which was needed for its fitness. Interestingly infection by the nematode was only successful if microbiota was present (compared to germ-free mice).
Furthermore, in a comprehensive analysis by Hahn et al. (2022) they showed the microbiome of sticklebacks changes with not just an infection of the cestodes parasite Schistocephalus solidus, but this is also dependent on the genotype of the parasite.
It is not only parasitic helminths that can alter the microbiome of their hosts. Koch and Schmid-Hempel (2011) showed the adaptive value of the microbiota of social bees, which can protect against parasites such as the trypanosomatid gut parasite Crithidia bombi. bacteria that could be used as probiotics to keep honeybees healthy.
Understanding the multidirectional interactions between parasites, microbiomes, and the host's immune system during infections could open new avenues of treatment and prevention (Reynolds et al., 2015).
Our results show a clear holobiont dysbiosis associated with a P.
hermaphrodita infection in the susceptible species D. reticulatum, but not in the resistant slug species A. valentianus, this dysbiosis is consistent with previous studies looking at host/parasite systems in vertebrates (Brealey et al., 2022;McKenney et al., 2015;Walk et al., 2010). We thank Tom Goddard and Jack Shepherd for useful discussions.

CONFLICT OF INTEREST STATEMENT
None declared.

DATA AVAILABILITY STATEMENT
Sequence data are available at the European Nucleotide Archive (ENA) under the ID PRJEB56262 (ERP141185): https://www.ebi.ac.

ETHICS STATEMENT
None required.